Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells

Abstract

Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) and PGC-1α-related coactivator (PRC). Suppression of PGC-1β and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1β, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

Keywords: cancer biology; cancer therapy; cell metabolism; cell signaling; cell surface receptor; drug resistance; insulin-like growth factor (IGF); mitochondria; mitophagy.

Figure 1.

IGF-1 induces mitochondrial biogenesis and expression of mitochondrial genes. A, RNA expression levels of PGC-1α, PGC-1β, and PRC in MCF-7, MDA-MB-231, and ZR75.1 cells cultured in complete medium were determined by qPCR, normalized to the housekeeping gene UBC, and presented as -fold change compared with MDA-MB-231 cells set at a value of 1. B, mitochondrial mass in MCF-7 and ZR75.1 cells. Cells were serum-starved for 4 h and stimulated with 10 ng/ml or 100 ng/ml IGF-1 for a further 20 h. Mitochondrial mass was measured by FACS using the MTG probe. C, immunofluorescence staining of TOM20 in MCF-7 cells following stimulation with IGF-1. MCF-7 cells were serum-starved for 4 h, followed by stimulation with 100 ng/ml IGF-1 for a further 20 h, after which they were prepared for immunofluorescence using an antibody specific for the mitochondrial outer membrane protein TOM20 (green). Nuclei were stained with Hoechst dye (blue), and actin was stained with phalloidin (red). The original magnification was ×40. Scale bars = 25 μm. D, expression levels of Aralar, PNC1, TFAM, and MFN1 mRNA in MCF-7 and ZR75.1 cells following stimulation with IGF-1. Cells were serum-starved for 4 h and stimulated with 10 ng/ml or 100 ng/ml IGF-1 for a further 20 h. The expression level of each gene was normalized to the housekeeping gene UBC and is shown as -fold change of the control (serum-starved cells) set at 1. E, qPCR showing the mRNA expression levels of PGC-1β and PRC in MCF-7 cells following stimulation with IGF-1. Cells were serum-starved for 4 h and stimulated with 10 ng/ml or 100 ng/ml IGF-1 for a further 4 h. Cells stimulated with 100 ng IGF-1 were treated with 20 μm LY294002 30 min prior to stimulation. The levels of PGC-1β and PRC mRNA were normalized to the housekeeping gene UBC and are presented as -fold change of the control (serum-starved) set at 1. In B, D, and E, data are presented from three independent experiments as mean ± S.E. Statistical analysis was performed using Student's t test (*, p < 0.05; **, p < 0.01).

Figure 2.

IGF-1 promotes mitochondrial biogenesis through induction of PGC-1β and PRC. A, expression of Aralar mRNA in MCF-7 cells following single or simultaneous suppression of PGC-1β and PRC. MCF-7 cells were transfected with PGC-1β or PRC siRNA for 48 h before being harvested for qPCR. Aralar expression levels were normalized to the housekeeping gene UBC and are shown as -fold change of the siRNA negative control (siNeg) set at 1. The data for this and all qPCR experiments are presented as mean ± S.E. from three independent experiments. B, expression of PGC-1β and PRC mRNA in MCF-7 cells following simultaneous suppression of PGC-1β and PRC. MCF-7 cells were transfected with PGC-1β or PRC siRNA for 48 h prior to serum starvation for 4 h and stimulation with 10 ng/ml or 100 ng/ml IGF-1 for a further 4 h. Gene expression was analyzed by qPCR, and mRNA levels were normalized to the housekeeping gene UBC and are presented as -fold change of the serum-starved controls set at 1. C, expression of Aralar, TFAM, and PNC1 mRNA in MCF-7 cells following simultaneous suppression of PGC-1β and PRC. MCF-7 cells were treated, and the results were analyzed as described in B. D, analysis of mitochondrial membrane potential by flow cytometry using the TMRE probe. MCF-7 cells were transfected with PGC-1β and PRC siRNA (gray) for 48 h compared with siNeg (black). A representative histogram of TMRE fluorescence is shown, with the scatter plot showing the geometric mean from three independent experiments. Statistical analysis using Student's t test indicated no significance. E, Western blot showing OXPHOS and VDAC1 levels in MCF-7 cells transfected with PGC-1β and PRC siRNA for 48 h. Cells were lysed and prepared for Western blotting with anti-OXPHOS, anti-VDAC1, and anti-α-tubulin antibodies. Quantification was performed by densitometry relative to α-tubulin and normalized to the siNeg control. The -fold change for VDAC1 is indicated below the Western blot. F, immunofluorescence of MCF-7 cells following transfection with PGC-1β and PRC siRNA for 72 h. The cells were stained with an antibody specific for the mitochondrial outer membrane protein TOM20 (white). Scale bars = 20 μm. The enlarged images below are six times larger. The number of rounded and reticular mitochondria was counted in a total of 100 fields per condition (10–20 cells/field) from three individual experiments and is presented in the bar chart as a percentage of total cells counted. G, levels of PRC and PG1-β mRNA suppression in MCF-7 cells used in D–F, determined by qPCR. In all panels, data are presented from three independent experiments as mean ± S.E. Statistical analysis was performed using Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.005).

Figure 3.

IGF-1R inhibition is associated with mitochondrial dysfunction. A, mitochondrial mass in MCF-7, ZR75.1, and MDA-MB-231 cells following exposure to BMS-754807 for 24 h. B, mitochondrial mass in MCF-7, ZR75.1 and MDA-MB-231 cells following exposure to LY294002 for 24 h. Mitochondrial mass was measured by FACS using the MTG probe. C, expression levels of PGC-1β and PRC in MCF-7 cells following exposure to BMS-754807 for 24 h. The levels of PGC-1β and PRC were determined by qPCR normalized to the housekeeping gene UBC and are shown as -fold change of the untreated control set at 1. D, representative Western blot showing the activity of the IGF-1R, PI3K, and MAPK signaling pathways, as measured by phosphorylation levels of IGF-1R, AKT, and ERK, respectively. MCF-7 and MCF-7-R cells were cultured in complete medium supplemented with or without 500 nm BMS-754807 (BMS), as indicated, followed by lysis and immunoblotting with anti-phospho and non-phospho antibodies against IGF-1R, ERK, and Akt. E, phospho-RTK array showing the phosphorylation levels of 42 different receptor tyrosine kinases in MCF-7 and MCF-7-R cells. The arrays were exposed to 100 μg of MCF-7 and MCF-7-R whole-cell lysates and probed with an HRP-conjugated pan phosphotyrosine antibody. F, EGFR expression in MCF-7 and MCF-7-R cells. MCF-7 and MCF-7-R cell lysates were prepared and analyzed by Western blotting using anti-EGFR and anti-β-actin antibodies. G, mitochondrial mass in MCF-7 and MCF-7-R cells, measured by flow cytometry using the MitoTracker Green probe. H, mitochondrial ROS in MCF-7 and MCF-7-R cells, measured by flow cytometry using the MitoSOX Red probe. I, expression levels of PGC-1β and PRC in MCF-7 and MCF-7-R cells. The levels of PGC-1β and PRC were determined by qPCR, normalized to the housekeeping gene UBC, and are presented as -fold change of the levels in MCF-7 cells set at 1. J, qPCR showing the levels of TFAM, Aralar, and PNC1 in MCF-7 compared with MCF-7-R cells. The expression level of each gene was normalized to the housekeeping gene UBC, and the levels in MCF-7-R cells are presented as -fold change of the levels in the MCF-7 cells set at 1. K, qPCR showing the expression levels of NFE2L2 in MCF-7 and MCF-7-R cells that were serum-starved and stimulated with IGF-1 or not. The levels of NFE2L2 were normalized to the housekeeping gene UBC and are shown as -fold change compared with serum-starved MCF-7 cells. Data are presented from three independent experiments as mean ± S.D. Statistical analysis was performed using Student's t test (*, p < 0.05; **, p < 0.01).

Figure 4.

The mitophagy mediators BNIP3 and BNIP3L are reduced in MCF-7-R cells. A, BNIP3 and BNIP3L expression in MCF-7 and MCF-7-R cells. The levels of BNIP3 and BNIP3L were determined by qPCR, normalized to the housekeeping gene UBC. The levels in MCF-7-R cells are presented as -fold change of the levels in the MCF-7 cells set at 1. B, BNIP3 protein expression in MCF-7 and MCF-7-R cells. MCF-7 cells were maintained in complete medium, whereas MCF-7-R cells were maintained in complete medium supplemented with 500 nm BMS-754807. Cells were lysed and prepared for immunoblotting using anti-BNIP3 and anti-β-actin antibodies. C, BNIP3 mRNA in MCF-7 and MCF-7-R cells under normoxic or hypoxic (1% O₂) conditions. Cells were exposed to hypoxia for 20 h. BNIP3 levels were determined by qPCR and normalized to the housekeeping gene UBC. The levels are presented as -fold change of expression in MCF-7 cells under normoxic conditions set at 1. D, BNIP3 protein expression in MCF-7 and MCF-7-R cells in normoxia (Norm), hypoxia (Hyp, 1% O₂), or reoxygenation (Reoxy). Cells were maintained under normoxia, exposed to hypoxia for 20 h, or exposed to hypoxia for 20 h followed by normoxia for a further 5 h. Cells were then lysed and prepared for Western blotting using anti-BNIP3 and anti-β-Actin antibodies. E, qPCR showing BNIP3L mRNA in MCF-7 and MCF-7-R cells under normoxic or hypoxic (1% O₂) conditions. Cells were exposed to hypoxia for 20 h. BNIP3L levels, normalized to UBC expression, are presented as -fold change relative to normoxia control samples for each cell line. F, LC3B levels in MCF-7 and MCF-7-R cells in the presence of 50 μm chloroquine (CQ) for 4 or 24 h. Cells were lysed and prepared for immunoblotting using anti-phospho-IGF-1R, anti-phospho-AKT, anti-AKT, anti-LC3B antibodies, and anti-α-tubulin antibodies. G, BNIP3 and BNIP3L expression in MCF-7 and MCF-7-R cells maintained in medium in the presence of 500 nm BMS-754807 or not, as indicated. The levels of BNIP3 and BNIP3L were normalized to the housekeeping gene UBC, and the levels are shown as -fold change of the levels in control (Ctrl) MCF-7 cells. H, PGC-1β and PRC expression in MCF-7 and MCF-7-R cells in the presence of 500 nm BMS-754807 or not, as indicated. PGC-1β and PRC levels, determined by qPCR, were normalized to the housekeeping gene UBC, and the levels are presented as -fold change of the levels in MCF-7 cells. The data for all PCR experiments are presented mean ± S.E. from three independent experiments. Statistical analysis was performed using Student's t test (*, p < 0.05; **, p < 0.01). I, LC3B protein levels in MCF-7 and MCF-7-R cells. MCF-7 and MCF-7-R cells were maintained in complete medium with or without 500 nm BMS-754807 as indicated. Cells were lysed and prepared for immunoblotting using anti-IGF-1R, anti-phospho-IGF-1R, anti-LC3B, and anti-β-actin antibodies. LCB3 expression was quantified by densitometry relative to actin and normalized to the MCF-7 cell controls. The -fold change in LC3B and LCB3II expression is indicated above and below the Western blot, respectively.

Figure 5.

BNIP3 is induced by IGF-1 in a PI3K-dependent manner. A, BNIP3 mRNA expression following stimulation with IGF-1 in MCF-7 cells. Cells were serum-starved for 4 h, stimulated with 10 ng/ml or 100 ng/ml IGF-1, and placed under normoxic or hypoxic conditions for a further 20 h. Cells stimulated with 100 ng/ml IGF-1 were pretreated with LY294002 or PD98059 prior to being stimulated and placed under hypoxic conditions. The levels of BNIP3 were determined by qPCR, normalized to the housekeeping gene UBC, and are presented as -fold change of the levels in the cells under serum-starved normoxic conditions. Data are presented as mean ± S.E. from three independent experiments. The graph represents the mean ± S.D. derived from three individual experiments. Statistical significance was determined using Student's t test (*, p < 0.05; **p < 0.01). B, BNIP3 protein expression following stimulation with IGF-1 in MCF-7 cells over time. Cells were serum-starved for 4 h and stimulated with 10 ng/ml IGF-1 from 4–24 h. β-Actin was used as a loading control. C, BNIP3 protein expression dependence on PI3K activity. Cells were serum-starved for 4 h and stimulated with 10 ng/ml IGF-1 for a further 20 h. LY294002 was added 30 min prior to IGF-1 stimulation. Cells lysates were probed with anti-phospho-IGF-1R, anti-phospho-AKT, anti-AKT, anti-BNIP3, and anti-β-actin antibodies. D, BNIP3 expression in MCF-7 cells following exposure to hypoxia (Hyp). Cells were maintained in complete medium and exposed to DMSO (−), LY294002, PD98059, or BMS-754807 and placed under hypoxia for 20 h. Cells lysates were prepared for immunoblotting using anti-BNIP3, anti-phospho-AKT, anti-AKT, anti-phospho-ERK and anti-ERK antibodies. Norm, normoxia. E, subcellular localization of BNIP3 in MCF-7 cells under normoxic or hypoxic conditions. Cells were serum-starved and stimulated with 100 ng/ml IGF-1 for 20 h Cells were then lysed and subjected to subcellular fractionation before being harvested for lysis and Western blotting with anti-BNIP3 and anti-VDAC antibodies. Cyto indicates cytoplasmic fraction, and mito indicates mitochondria-enriched fraction. F, BNIP3 expression following IGF-1 stimulation in the presence of Myc inhibitor. MCF-7 cells were serum-starved for 4 h and stimulated with 100 ng/ml IGF-1 in the presence or absence of 2-MeOE2 as indicated. Cell lysates were prepared for immunoblotting using anti-BNIP3, anti-phospho-AKT, anti-AKT, and anti-β-actin antibodies. For all Western blots, a representative is presented from a minimum of three independent experiments that showed similar results.

Figure 6.

MCF-7-R cells display reduced levels of mitophagy, reduced mitochondrial activity, and increased sensitivity to glycolysis inhibition. A, localization of BNIP3 at mitochondria of MCF-7 and MCF-7-R cells. Cells were exposed to hypoxia for 24 h and prepared for immunofluorescence with an antibody specific for BNIP3 (red) and a TOM20 antibody (green) as a mitochondrial marker. Co-localization, shown in white, was assessed using the Colocalization plugin for ImageJ described under “Materials and Methods.” B, immunofluorescence depicting levels of mitophagy in MCF-7 and MCF-7-R cells. Cells were exposed to hypoxia for 24 h and then fixed and stained for TOM20 (green) or phalloidin (actin, red). Nuclei were stained with Hoechst (blue). The graph shows quantification of the number of cells that were undergoing mitophagy following exposure to hypoxia as described in the text. The graph represents the average ± S.E. of three individual experiments. Statistical significance was determined using Student's t test (*, p < 0.05; **, p < 0.01). C, co-localization of TOM20 with LAMP1 in MCF-7 and MCF-7-R cells. Cells were exposed to hypoxia for 24 h, and then they were fixed and stained with anti-OM20 (green) or -LAMP1 (red) antibodies. D, mitochondrial stress test showing mitochondrial activity in MCF-7 and MCF-7-R cells. The top plot shows the OCR, measured using a Seahorse XFp analyzer, over a course of 2 h under basal conditions and following addition of the indicated uncouplers. The bar chart shows basal respiration and ATP production, which were calculated as described under “Materials and Methods.” The data represent the mean ± S.E. derived from three independent experiments. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. E, growth rates of MCF-7 parental and MCF-7-R cells following exposure to 25 mm 2-DG. Cells were initially plated at a density of 2.5 × 10⁴/well of a 24-well plate, and cell density was assessed every 24 h using crystal violet staining and quantified using Odyssey scanning. Representative cell culture plates are shown in the top panel, and cell density from three independent experiments are shown in the graph as mean ± S.D. Statistical analysis for each experiment was performed using Student's t test (*, p < 0.05; **, p < 0.01). Scale bars = 25 μm.